title: Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.
abstract: DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.
authors.affiliation: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Obstetrics and Gynecology and Center for Reproductive Sciences, University of California San Francisco, San Francisco, California 94143, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Blaschke
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Ebata
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Karimi
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Zepeda-Martínez
authors.forename: Preeti
authors.initials: P
authors.lastname: Goyal
authors.forename: Sahasransu
authors.initials: S
authors.lastname: Mahapatra
authors.forename: Angela
authors.initials: A
authors.lastname: Tam
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Laird
authors.forename: Martin
authors.initials: M
authors.lastname: Hirst
authors.forename: Anjana
authors.initials: A
authors.lastname: Rao
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Lorincz
authors.forename: Miguel
authors.initials: M
authors.lastname: Ramalho-Santos
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D001755:Blastocyst / Q000378:metabolism; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D055786:Gene Knockout Techniques; D051379:Mice; D011485:Protein Binding / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000975:Antioxidants; D003470:Culture Media; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C540839:TET1 protein, mouse; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D049308:Dioxygenases; C551914:Tet2 protein, mouse; D001205:Ascorbic Acid